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Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

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Thermal unfolding curves of ligases #6, #7 and 10C.Thermal unfolding was monitored by circular dichroism at 222 nm. For each measurement 10 accumulations were acquired.
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pone-0112028-g005: Thermal unfolding curves of ligases #6, #7 and 10C.Thermal unfolding was monitored by circular dichroism at 222 nm. For each measurement 10 accumulations were acquired.

Mentions: In order to assess if the unique enzymatic activity of ligase 10C at 65°C was correlated to increased structural stability, we measured thermal denaturation curves of all three ligases by circular dichroism. In preparation of the thermal unfolding experiment, we measured the CD spectra of the three enzymes (Figure S4). All three spectra exhibited two minima of negative ellipticity at 205 nm and between 220 and 225 nm, respectively. While those minima suggested α-helical secondary structural content [24], the 205 nm minimum was substantially more negative than the second minimum, which differs from purely alpha helical proteins that have similar absolute values for both minima. In fact, the three-dimensional structure of ligase 10C recently solved by NMR revealed that α-helices and β-strand regions are essentially absent in ligase 10C [20]. Nevertheless, we used the strong negative ellipticity of all three ligases at 222 nm to monitor thermal unfolding of the proteins over a temperature range from 5 to 91°C. We found all three enzymes to give the characteristic single sigmoidal transition corresponding to a two-state unfolding reaction (Figure 5). As determined from the curves, the enzymes showed very different melting temperatures. Ligase 10C had the highest melting temperature (Tm = 72°C), which was 35 degrees higher than the Tm of ligase #6 (37°C), and 24 degrees higher than the Tm of ligase #7 (48°C). The high melting temperature of 72°C for ligase 10C was in agreement with its retained enzymatic activity at 65°C as the enzyme had not undergone unfolding yet. In contrast, ligases #6 and #7 were fully denatured at 65°C, and, therefore, their complete lack of enzymatic activity at 65°C could be explained by their unfolding.


Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Thermal unfolding curves of ligases #6, #7 and 10C.Thermal unfolding was monitored by circular dichroism at 222 nm. For each measurement 10 accumulations were acquired.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230948&req=5

pone-0112028-g005: Thermal unfolding curves of ligases #6, #7 and 10C.Thermal unfolding was monitored by circular dichroism at 222 nm. For each measurement 10 accumulations were acquired.
Mentions: In order to assess if the unique enzymatic activity of ligase 10C at 65°C was correlated to increased structural stability, we measured thermal denaturation curves of all three ligases by circular dichroism. In preparation of the thermal unfolding experiment, we measured the CD spectra of the three enzymes (Figure S4). All three spectra exhibited two minima of negative ellipticity at 205 nm and between 220 and 225 nm, respectively. While those minima suggested α-helical secondary structural content [24], the 205 nm minimum was substantially more negative than the second minimum, which differs from purely alpha helical proteins that have similar absolute values for both minima. In fact, the three-dimensional structure of ligase 10C recently solved by NMR revealed that α-helices and β-strand regions are essentially absent in ligase 10C [20]. Nevertheless, we used the strong negative ellipticity of all three ligases at 222 nm to monitor thermal unfolding of the proteins over a temperature range from 5 to 91°C. We found all three enzymes to give the characteristic single sigmoidal transition corresponding to a two-state unfolding reaction (Figure 5). As determined from the curves, the enzymes showed very different melting temperatures. Ligase 10C had the highest melting temperature (Tm = 72°C), which was 35 degrees higher than the Tm of ligase #6 (37°C), and 24 degrees higher than the Tm of ligase #7 (48°C). The high melting temperature of 72°C for ligase 10C was in agreement with its retained enzymatic activity at 65°C as the enzyme had not undergone unfolding yet. In contrast, ligases #6 and #7 were fully denatured at 65°C, and, therefore, their complete lack of enzymatic activity at 65°C could be explained by their unfolding.

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

Show MeSH